LONG-TERM GOAL: to characterize the events/mechanisms of the adipocyte differentiation program and verify these events in physiological context. The immediate goal is to understand "mitotic clonal expansion" (MCE), which is required for adipogenesis. The well-characterized 3T3-L1 preadipocyte --) adipocyte differentiation system will serve as primary model. Wild-type and mutant/knockout mouse embryo fibroblasts (MEFs) and a preadipocyte "in vivo implantation" system will test ex vivo results in physiological context. SPECIFIC AIMS: to determine the: 1) roles of C/EBPbeta/delta and Calpain: C/EBPbeta/delta lack DNA binding activity early in the program. After a long lag this function is acquired concomitant with phosphorylation and release from inhibitory constraint. As the cells synchronously enter S-phase of MCE. Once C/EBPbeta/delta acquire DNA binding activity, they transcriptionally activate the C/EBPalpha and PPARgamma genes which transcriptionally activate genes that produce the adipocyte phenotype. Relevant phosphorylation sites on C/EBPbeta/delta will be identified, the responsible kinase(s) and interaction partners identified and effects on C/EBPbeta/delta function determined. MCE will be characterized along with converging events at the G1-S checkpoint that are responsible for progression of MCE and differentiation. Among these is the activation of Calpain which we showed plays a crucial role in initiating p27/KIP1 turnover, an essential event in the activation of Cdk2-cyclinA/entry of S-phase. The mechanism(s) by which calpain activation is triggered and its possible role in the transcriptionally activating the Cdk2 gene, a unique feature of adipogenic MCE, will be explored, and 2) roles of CUP/AP-2alpha and Spl: Previous studies identified repressive regulatory elements for, i.e. CUP/AP-2(_and Spl, in the C/EBP( gene promoter. Down-regulation of these repressors occurs concomitant with the onset of MCE just prior to the transcriptional activation of the C/EBPalpha gene. The mechanisms by which these repressors are down-regulated and the effects of forced expression and "knockdown" on MCE/differentiation and/or de-differentiation will be determined. The 32kDa CUP/AP-2alpha- interacting protein, p32, previously identified in our lab, will be characterized and its role determined.